Regulation of skeletal myogenesis by Notch.

Notch signaling has emerged as a key player in skeletal muscle development and regeneration. Simply stated, Notch signaling inhibits differentiation. Accordingly, fine-tuning the pathway is essential for proper muscle homeostasis. This review will address various aspects of Notch signaling, including our current views of the core pathway, its effects in muscle, its interactions with other signaling pathways, and its relationship with ageing.

The Notch effector Hey1 associates with myogenic target genes to repress myogenesis.

Members of the Hey family of transcriptional repressors are basic helix-loop-helix proteins that are thought to act downstream of Notch in diverse tissues. Although forced expression of Hey1, a target of Notch in myoblasts, is sufficient to recapitulate inhibitory effects of the pathway on differentiation, how Hey1 interferes with myogenic transcription has not been fully elucidated. We provide multiple lines of evidence that Hey1 does not target the intrinsic transcriptional activity of the skeletal muscle master regulator MyoD. Our results indicate instead that Hey1 is recruited to the promoter regions of myogenin and Mef2C, two genes whose induction is critical for myogenesis. Expression of Hey1 in C2C12 myoblasts correlates with reduced recruitment of MyoD to these promoters, arguing that Hey1 inhibits myogenesis by associating with and repressing expression of key myogenic targets.

Inhibition of myogenesis by Notch: evidence for multiple pathways.

Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand-mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA-mediated knockdown of Hey1 had no effect on Notch's ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch-responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA-mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR.

Functional analysis of Hes-1 in preadipocytes.

Notch signaling blocks differentiation of 3T3-L1 preadipocytes, and this can be mimicked by constitutive expression of the Notch target gene Hes-1. Although considered initially to function only as a repressor, recent evidence indicates that Hes-1 can also activate transcription. We show here that the domains of Hes-1 needed to block adipogenesis coincide with those necessary for transcriptional repression. HRT1, another basic-helix-loop-helix protein and potential Hes-1 partner, was also induced by Notch in 3T3-L1 cells but did not block adipogenesis, suggesting that Hes-1 functions primarily as a homodimer or possibly as a heterodimer with an unknown partner. Purification of Hes-1 identified the Groucho/transducin-like enhancer of split family of corepressors as the only significant Hes-1 interacting proteins in vivo. An evaluation of global gene expression in preadipocytes identified approximately 200 Hes-1-responsive genes comprising roughly equal numbers of up-regulated and down-regulated genes. However, promoter analyses indicated that the down-regulated genes were significantly more likely to contain Hes-1 binding sites, indicating that Hes-1 is more likely to repress transcription of its direct targets. We conclude that Notch most likely blocks adipogenesis through the induction of Hes-1 homodimers, which repress transcription of key target genes.

Inhibition of EBF function by active Notch signaling reveals a novel regulatory pathway in early B-cell development.

The Notch signaling pathway is involved in several lineage commitment and differentiation events. One of these is fate determination of the common lymphoid progenitor, promoting T-cell development at the expense of B-cell differentiation. It has been suggested that this process relies on Notch's ability to inhibit E proteins, which are crucial for early B-cell development. Here, we report that Notch signaling also modulates the function of the transcription factor, early B-cell factor (EBF). Transient transfection of intracellular Notch1 (Notch1-IC) into a pre-B cell line resulted in the down-regulation of EBF-regulated promoters and diminished the capacity of EBF to activate these promoters in an epithelial cell line. This correlated with a reduction in the ability of EBF to bind DNA. Ligand-induced stimulation of endogenous Notch receptors with Delta4 mimicked the activity of Notch1-IC toward EBF. These data suggest that Notch signaling may affect B-versus T-lineage commitment by the targeting of both EBF and E2A.

Notch signaling: the demise of elegant simplicity.

Notch signaling can be viewed as an elegantly simple pathway that begins when the Notch receptor binds ligand, and ends when the Notch intracellular domain enters the nucleus and activates transcription. However, it is becoming increasingly clear that this core pathway is subject to a wide array of regulatory influences, from those that affect ligand-receptor interactions to those that govern the choice of Notch target genes. Even Notch ligands are now being scrutinized with respect to the possibility that they, too, function in the nucleus. A complete understanding of Notch signaling therefore requires us to look well beyond the core pathway.

Consequences of Notch-mediated induction of Jagged1.

Notch signaling is initiated upon contact of cells expressing Notch receptors with those expressing ligands. While examining the dynamic response of NIH 3T3 cells to cells expressing the Notch ligand Jagged1, we found that Notch signaling resulted in increased levels of the ligand Jagged1. Induction of Jagged1 was delayed relative to the generation of active Notch and dependent on the transcription factor p63. The induced Jagged1 had no apparent autocrine effects on Notch signaling but could promote signaling in naïve cells. These results describe a mechanism through which Notch signaling can be relayed from cell to cell.

Dual roles for the Notch target gene Hes-1 in the differentiation of 3T3-L1 preadipocytes.

The process of adipogenesis involves a complex program of gene expression that includes down-regulation of the gene encoding Hes-1, a target of the Notch signaling pathway. To determine if Notch signaling affects adipogenesis, we exposed 3T3-L1 preadipocytes to the Notch ligand Jagged1 and found that differentiation was significantly reduced. This effect could be mimicked by constitutive expression of Hes-1. The block was associated with a complete loss of C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma) induction and could be overcome by retroviral expression of either C/EBPalpha or PPARgamma2. Surprisingly, small interfering RNA (siRNA)-mediated reduction of Hes-1 mRNA in 3T3-L1 cells also inhibited differentiation, suggesting an additional, obligatory role for Hes-1 in adipogenesis. This role may be related to our observation that both Notch signaling and Hes-1 down-regulate transcription of the gene encoding DLK/Pref-1, a protein known to inhibit differentiation of 3T3-L1 cells. The results presented in this study establish a new target downstream of the Notch-Hes-1 pathway and suggest a dual role for Hes-1 in adipocyte development.

Notch and Schwann cell transformation.

Benign plexiform neurofibromas in NF1 patients can transform spontaneously into malignant peripheral nerve sheath tumors (MPNSTs). Although mutations in the p53 gene have been found in a subset of MPNSTs and mouse models support a role for p53 mutations in malignant conversion, we found that each of three Schwann cell lines derived from human MPNSTs possessed active p53. One of the lines expressed the Notch intracellular domain (NICD), indicative of ongoing Notch signaling. Consistent with a role in malignancy, NICD was able to transform primary rat Schwann cells. Transformation was robust--NICD-transduced cells generated tumors in nude rats--and was associated with the loss of markers associated with Schwann cell differentiation. These data suggest that aberrant Notch signaling may contribute to the conversion of benign neurofibromas to MPNSTs.

The intracellular form of notch blocks transforming growth factor beta-mediated growth arrest in Mv1Lu epithelial cells.

Notch signaling influences a variety of cell fate decisions during development, and constitutive activation of the pathway can provoke unbridled cell growth and cancer. The mechanisms by which Notch affects cell growth are not well established. We describe here a novel link between Notch and cell cycle control. We found that Mv1Lu epithelial cells harboring an oncogenic form of Notch (NICD) are resistant to the cell cycle-inhibitory effects of transforming growth factor beta (TGF-beta). NICD did not affect TGF-beta signaling per se but blocked induction of the Cdk inhibitor p15(INK4B). c-Myc, whose down-regulation by TGF-beta is required for p15(INK4B) induction, remained elevated in the NICD-expressing cells. c-Myc expression was also maintained in low serum, indicating that Notch's effects on c-Myc are not specific to TGF-beta. Our results are consistent with a model in which a strong Notch signal indirectly deregulates c-Myc expression and thereby renders Mv1Lu epithelial cells resistant to growth-inhibitory signals.

Inhibition of p300/CBP by early B-cell factor.

Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development. It activates transcription of several B-cell-specific genes, including the lambda5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor. In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP. We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo. Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor. In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the lambda5 gene. This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro. However, such a repressed complex is not likely to form at the lambda5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A. Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during lambda5 promoter activation.